ABSTRACT Owing to the adverse impact of vitamin D deficiency on the skeleton and risk of skin cancer associated with prolonged cutaneous exposure to sunlight, oral supplementation with vitamin D3 or D2 has been widely accepted and promoted as the mode of achieving and maintaining calcium homeostasis in children and adults. Given recent appreciation of the profound affect that the gut microbiome has on host human health and disease, with support of this supplement we will be performing the first pilot study to compare and contrast the impact of the most widely used oral vitamin D supplement (vitamin D3) versus the more rapidly acting, experimental 25-hydroxylated metabolite, 25D3, on the gut-residing microbes that have the first opportunity to encounter and respond to those vitamin D metabolites. With support from this supplement, we wish to take advantage of the still-recruiting, multi-ethnic vitamin D replacement trial comparing oral administration of vitamin D3 and 25-hydroxyvitamin D3 (25D3) in vitamin D deficient subjects (total serum 25D ?20 ng/ml) set forth in Specific Aim 3 of the parent grant R01AR063910. This application includes a plan for supplemental analysis of the gut microbiome in those subjects before and after normalization of total and free serum 25D. We propose that i) there will exist predictable changes in the taxomic 16S rRNA-sequencing profiles of the stool microbiota and 2) the impact of that altered bacterial microbiome on the host's skeletal and immune homeostasis that will be conditioned by the form of vitamin D supplementation and the host's ethnicity (White, Black, Hispanic, Asian). These new analyses will i) compliment the already-funded program's novel assay technology for determining serum free 25D and 1,25D and ii) further advance our definition of `personalized' vitamin D health in terms of both classical (bone) and non-classical (immune) actions of vitamin D. !